1. Field of the Invention
The present invention relates to the synthesis of polynucleotides using solid phase synthesis techniques.
2. Description of the Related Art
A polynucleotide is a linear chain of nucleotides, where nucleotides are composed of a sugar, a base and a phosphate. The sugar of one nucleotide joins to the phosphate of the adjacent nucleotide in order to form the chain. A human gene is formed from a polynucleotide wherein the sugar is deoxyribose, and the base at each position in the chain is selected from adenine (A), cytosine (C), guanine (G) and thymine (T). Thus, the only chemical difference between links in a chain of polynucleotides is the base present in the link. In large part, genes differ from one another due to their different chain lengths and due to having a different sequence of the four bases along the chain.
Much of the discovery research in pharmaceutical companies is focused on genes, either as targets for drug development or as protein therapeutics. Due to recent and continuing efforts directed to determining the sequence of genomic DNA molecules, these companies have access to the base sequence a majority of the human genes. In fact, these companies are overwhelmed with potential opportunities, acutely aware that their competitors are looking at the same set of possibilities, and currently unable to work on more than a fraction of the genes that have been identified.
One of the major bottlenecks in performing research in the area of genes is the time and effort required to prepare genes of a desired base sequence which can be used, e.g., as a research or diagnostic tool, or a potential therapeutic agent. Genes are typically several hundred to a few thousand nucleotides long, and are defined by their nucleotide sequence. In order to successfully prepare a gene, the nucleotides of the gene must be in a precisely specified order, i.e., when a particular one of the four bases is specified to be at a particular location along the chain, then that base and no other base must be present at that particular location—there cannot be any extra or missing nucleotide bases.
The manufacture of short chains of nucleotides, often referred to as oligonucleotides when the chains are less than about 100 nucleotides in length, is a well developed and commercially employed process. Typically, an insoluble support is joined to a first nucleotide, and the support is placed into a solution with a reactive precursor of the second nucleotide. After the second nucleotide has been joined to the first nucleotide, the reaction mixture is washed through a frit, thus separating the solid support from any unreactive precursor. The solid phase containing the nascent oligonucleotide is then exposed to the reactive precursor of the third nucleotide, and this procedure is followed repetitively until the desired oligonucleotide is prepared.
Not all of the nascent oligonucleotide reacts as desired with a reactive precursor, and so even though the chemical yield is typically greater than 90%, the yield of desired product, based on the amount of first nucleotide bound to solid support, drops dramatically as the number of repetitive cycles increases. Also, it is empirically observed that some oligonucleotides made by this process, even when they contain the desired number of nucleotides, do not have precisely the desired base sequence. Perhaps a particular nucleotide precursor will occasionally react twice with a growing chain, or perhaps all of a particular nucleotide precursor is not washed away after a round of reaction and remains to compete with the next addition of nucleotide precursor. In any event, this approach to making polynucleotides has not proved effective at making chains of defined sequence having over about 100 nucleotides in length. See the following references for discussion of making oligonucleotides: Schmitz, C. and Reetz, M. T., Org. Lett. 1(11):1729-1731 (1999);
In part because genes are such a desirable research tool, and even potential therapeutic agents, several research groups have directed attention to finding ways to prepare polynucleotides, and particularly genes, having many hundred bases in a pre-defined sequence, and have published results from their studies. See, e.g., Pachuk, C. J. et al., Gene 243:19-25 (2000); Evans, G. A., PCT International Publication No. WO99/14318 (1999): Hunkapiller, M. W. et al., U.S. Pat. No. 5,942,609 issued August 1999; Dietrich, R. et al., Biotech. Tech. 12(1):49-54 (1998); Rosenblum, M. G. et al., J. Interferon and Cytokine Res. 15:547-555 (1995); Kato, T. et al., Anal. Biochem. 220:428-429 (1994); St{dot over (a)}hl, S. et al. BioTechniques 14(3):424-434 (1993); Dombrowski, K. E. and Wright, S. E., Nucl. Acids Res. 20(24):6743-6744 (1992); Makarova, K. S. et al. Mol. Bio. (Mosk) 26(1):93-103 (1992); Beattie, K. L. and Fowler, R. F., Nature 352:548-549, 742 (1991); Filippov, S. A. et al., Bioorg. Khim. 16(8):1045-1051 (1990); Beattie, K. L. et al., Biotechnol. Appl. Biochem. 10:510-521 (1988); and Jerala R. and Turk V, Nuc. Acids Res. 16(5):1759-1766 (1988); Hostomsk?, Z. et al., Nuc. Acids Res. 15(12):4849-4856 (1987).
Each of the approaches described in these publications has strengths and weaknesses. But none of these approaches has proved entirely successful in the efficient production of polynucleotides of pre-defined sequence having several hundred nucleotides. For instance, in one approach, genes or other large DNA fragments are synthesized by using the enzyme DNA ligase to join short, chemically-synthesized fragments of DNA into longer fragments. A few oligonucleotide fragments at a time are allowed to anneal under conditions that favor formation of correct, double-stranded fragments. These fragments are mixed with adjacent fragments in the target sequence and subjected to enzymatic DNA ligation reactions. The order in which the fragments assemble is determined by single-stranded overhangs at the end of each short fragments. One principal factor limiting the reliability of gene synthesis by this approach is the low fidelity of the DNA ligase reaction: the enzyme can join fragments with mismatched ends and thus assemble the fragments in the wrong order. Several variations on this basic strategy have been described (see, e.g., Khorana, H., Science 203:614-625 (1979); Stabinsky, Y., U.S. Pat. No. 4,652,639; and Hostomsky, Z. and J. Smrt, Nucleic Acids Symposium Series (18):241-244 (1987). Some of these strategies involve the use of polymerase to produce some or all of the product that is cloned (see, e.g., Withers Martinez C. et al. Protein Engineering 12:1113-20 (1999) and U.S. Pat. No. 5,492,609 to Hunkapiller and Hiatt). Other approaches have other shortcomings.
Because DNA is at the heart of modern biology, reliable and cost-effective gene synthesis has the potential to play a part in moving biology towards an engineering approach to product development rather than a purely discovery-based approach. More specifically, cost-effective gene synthesis has the potential to save drug discovery researchers hundreds of millions of dollars by allowing them to outsource complex molecular biology projects. As described in detail below, the present invention provides a significant advancement in the preparation of polynucleotides such as genes.